Fig 2: Determination of soluble TAM receptors and Gas6 levels in synovial fluid of osteoarthritis patients. Soluble Axl (sAxl), soluble Mer (sMer), soluble Tyro3 (sTyro3), and Gas6 levels were determined in synovial fluids of OA patients (n = 12) by ELISA (A). The correlation between sAxl and Gas6 in synovial fluid was evaluated by Pearson’s coefficients (B). Results are shown as the mean ± SEM. * p < 0.05 and *** p < 0.001 when comparing sAxl with sMer, sTyro3, and Gas6. ## p < 0.01 and ### p < 0.001 when comparing sMer versus sTyro3, and sTyro3 versus Gas6, respectively; p values were determined by ANOVA with post hoc Tukey’s test (multiple groups).

Fig 3: C-10 fails to protect against ADRN in Tyro3-knockout mice.(A) Urinary albumin/creatinine ratio in Tyro3–wild-type (WT) and -knockout (KO) mice with ADRN treated with vehicle or C-10. (B) Twelve-hour albumin excretion in control and ADRN mice. (C) Periodic acid–Schiff–stained kidney images at ×200 (top) and ×400 (bottom) magnifications. Scale bars: 50 μm. (D) Glomerulosclerosis scoring in control and ADRN mice (n = 6 mice per group). *P < 0.05, ***P < 0.001, ****P < 0.0001 compared with WT ADR+Vehicle mice; #P < 0.05, ####P < 0.0001 compared with Tyro3-KO ADR+C-10 mice by 1-way ANOVA with Bonferroni’s correction.

Fig 4: C-10 is a TYRO3 agonist.(A) Cultured human podocytes were treated with control vehicle (C) or TYRO3 agonists (compounds 1–12) for 30 minutes. Cell lysates were probed for phosphorylated or total TYRO3 (p-TYRO3 or t-TYRO3). GAPDH was used as a loading control. (B) Cultured human podocytes were stimulated with compound 10 (C-10) for 30 minutes at different doses as indicated, and cell lysates were probed for p-TYRO3 or t-TYRO3 and for p-AKT or t-AKT. GAPDH was used as a loading control. (C) Cultured podocytes were treated with vehicle control (C), 10 μM TNF-α alone (T), or TNF-α with TYRO3 agonists (compounds 1–12) for 2 hours. Real-time PCR analysis was performed for expression of NF-κB target genes, IL6 and CCL2 (n = 3). *P < 0.05, **P < 0.01 compared with TNF-α–treated cells by 1-way ANOVA with Bonferroni’s correction.

Fig 5: C-10 is a selective agonist of TYRO3.(A) Cultured human podocytes were stimulated with C-10 at different doses for 30 minutes. Cell lysates were probed with an antibody recognizing the phosphorylated form of all 3 TAM receptors. Phosphorylated MER, AXL, and TYRO3 receptors are differentiated by their corresponding molecular weight. (B) Immortalized podocytes were transduced with either control vector or lentiviral vectors expressing individual FLAG-tagged TAM receptors (AXL-FLAG, TYRO3-FLAG, or MER-FLAG). Lysates from transduced cells were probed for FLAG-tagged protein expression. (C) Immortalized podocytes from B were treated with 100 nM C-10 for 30 minutes, and lysates were probed for phosphorylated or total AKT (p-AKT or t-AKT) and FLAG proteins. GAPDH was used as a loading control. Representative blots of 3 independent experiments are shown. (D) Drug affinity responsive target stability (DARTS) assay was performed to test the direct binding of C-10 to TYRO3. Podocyte lysates were preincubated with various concentrations of C-10 as indicated at 25°C for 1 hour prior to digestion with Pronase (0 or 1:1000 dilution) for 20 minutes. Lysates were then probed for TYRO3 expression, with GAPDH as a loading control.